Activation of Toll-like receptors induces dimerization and the recruitment of the death domain (DD) adaptor protein MyD88 into an oligomeric post receptor complex termed the Myddosome. The Myddosome is a hub for inflammatory and oncogenic signaling and has a hierarchical arrangement with 6&ndash;8 MyD88 molecules assembling with exactly 4 of IRAK-4 and 4 of IRAK-2. Here we show that a conserved motif in IRAK-4 (Ser8-X-X-X-Arg12) is autophosphorylated and that the phosphorylated DD is unable to form Myddosomes. Furthermore a mutant DD with the phospho-mimetic residue Asp at this position is impaired in both signalling and Myddosome assembly. IRAK-4 Arg12 is also essential for Myddosome assembly and signalling and we propose that phosphorylated Ser8 induces the N-terminal loop to fold into an &alpha;-helix. This conformer is stabilised by an electrostatic interaction between phospho-Ser8 and Arg12 and would destabilise a critical interface between IRAK-4 and MyD88. Interestingly IRAK-2 does not conserve this motif and has an alternative interface in the Myddosome that requires Arg67, a residue conserved in paralogues, IRAK-1 and 3(M).

f4: Reciprocal pull down assays confirm inability of Ser8-P death domain to interact with MyD88.(A) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit polyclonal anti-human MyD88 death domain antibody. Lane 1 to 3 are control lanes each loaded with 3 μg of the protein samples: lane 1-MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 6 were loaded with the pull down experiment samples as indicated. (B) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit monoclonal anti-human IRAK-4 death domain antibody. Lane 1 to 3 were control lanes each loaded with 3 μg of the protein samples: lane 1 corresponded to MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 7 were loaded with the pull down experiment samples as indicated. One repeat of three biological replicates is shown.

Mentions:
To confirm this conclusion, pull down experiments were carried out. MyD88 DD was incubated with either IRAK-4 DD or Ser 8 phosphorylated IRAK-4 DD at a final concentration of 0.5 mg.ml−1 overnight at room temperature. A rabbit polyclonal antibody specific for human MyD88 DD and goat anti-rabbit IgG magnetic beads were used to precipitate MyD88 protein complexes. After three washes the samples were analysed by SDS-PAGE to identify the complexes formed. The results show that only the non-phosphorylated IRAK-4 DD was able to bind MyD88 DD (Fig. 4A, lane 5). In reciprocal pull down experiments an antibody directed at the IRAK-4 N-terminal was used. This experiment confirms that only non-phosphorylated IRAK-4 DD was able to bind MyD88 DD (Fig. 4B, lane 7).

f4: Reciprocal pull down assays confirm inability of Ser8-P death domain to interact with MyD88.(A) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit polyclonal anti-human MyD88 death domain antibody. Lane 1 to 3 are control lanes each loaded with 3 μg of the protein samples: lane 1-MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 6 were loaded with the pull down experiment samples as indicated. (B) 4–20% reducing Tris-Glycine SDS PAGE of samples precipitated using a rabbit monoclonal anti-human IRAK-4 death domain antibody. Lane 1 to 3 were control lanes each loaded with 3 μg of the protein samples: lane 1 corresponded to MyD88 DD, lane 2-non-phosphorylated IRAK-4 death domain, lane 3-Ser8 phosphorylated IRAK-4 death domain. Lanes 4 to 7 were loaded with the pull down experiment samples as indicated. One repeat of three biological replicates is shown.

Mentions:
To confirm this conclusion, pull down experiments were carried out. MyD88 DD was incubated with either IRAK-4 DD or Ser 8 phosphorylated IRAK-4 DD at a final concentration of 0.5 mg.ml−1 overnight at room temperature. A rabbit polyclonal antibody specific for human MyD88 DD and goat anti-rabbit IgG magnetic beads were used to precipitate MyD88 protein complexes. After three washes the samples were analysed by SDS-PAGE to identify the complexes formed. The results show that only the non-phosphorylated IRAK-4 DD was able to bind MyD88 DD (Fig. 4A, lane 5). In reciprocal pull down experiments an antibody directed at the IRAK-4 N-terminal was used. This experiment confirms that only non-phosphorylated IRAK-4 DD was able to bind MyD88 DD (Fig. 4B, lane 7).

Activation of Toll-like receptors induces dimerization and the recruitment of the death domain (DD) adaptor protein MyD88 into an oligomeric post receptor complex termed the Myddosome. The Myddosome is a hub for inflammatory and oncogenic signaling and has a hierarchical arrangement with 6&ndash;8 MyD88 molecules assembling with exactly 4 of IRAK-4 and 4 of IRAK-2. Here we show that a conserved motif in IRAK-4 (Ser8-X-X-X-Arg12) is autophosphorylated and that the phosphorylated DD is unable to form Myddosomes. Furthermore a mutant DD with the phospho-mimetic residue Asp at this position is impaired in both signalling and Myddosome assembly. IRAK-4 Arg12 is also essential for Myddosome assembly and signalling and we propose that phosphorylated Ser8 induces the N-terminal loop to fold into an &alpha;-helix. This conformer is stabilised by an electrostatic interaction between phospho-Ser8 and Arg12 and would destabilise a critical interface between IRAK-4 and MyD88. Interestingly IRAK-2 does not conserve this motif and has an alternative interface in the Myddosome that requires Arg67, a residue conserved in paralogues, IRAK-1 and 3(M).